Antibodies have proven useful in medical applications for both diagnosis and therapy, and in biotechnology applications including cell separation. More generally, their high degree of binding specificity facilitates their use in the identification and localization of any compound to which antibodies can be generated in conjunction with techniques as varied as electron microscopy and enzyme linked immunosorbent assays.
Antibodies are comprised of both heavy and light chain polypeptides joined by interchain disulfide bonds and other intramolecular interactions. An individual heavy chain is paired with an individual light chain by these disulfide bonds. Of the different classes or isotypes of antibodies, three isotypes (IgD, IgE, and IgG) are comprised of two identical heavy chain/light chain pairs joined by a disulfide bond, and the remaining two isotypes (IgA and IgM) are comprised of more complicated polymers of identical heavy chain/light chain pairs. Each chain contains a constant region and a variable region. The constant regions are peculiar to the animal that generates the antibody and the specific isotype of antibody, while the variable regions conform to the structure of the epitope to which the antibody binds.
The term “antigen” is used herein to refer to a substance, whether an entire molecule or a domain within a molecule, which is capable of eliciting production of antibodies with binding specificity to that substance. Further, the term antigen is applied herein to substances, which in wild type host organisms would not elicit antibody production by virtue of self-recognition, but can elicit such a response in a host animal with the appropriate genetic engineering.
The term “epitope” is used herein to refer to the discrete, three-dimensional sites on an antigen, which are recognized by B lymphocytes. Epitopes are the immunologically active regions on a complex antigen, the regions that actually bind to a B-cell receptor, and that are actually bound by the resulting antibody molecules that are produced by the B-cell. Antigens generally contain at least one epitope and usually more than one epitope. Epitopes on protein antigens can be linear or non-linear. Linear epitopes are those comprised of contiguous amino acid residues in the amino acid sequence of a protein. Linear epitopes may or may not require conformational folding to form the native three-dimensional structure and elicit an immune response that produces antibodies with binding specificity to the antigen. Non-linear epitopes are comprised of non-contiguous amino acid residues. Thus, non-linear epitopes always require some degree of protein folding to bring the requisite amino acid residues into the proximity of one another to form the native three-dimensional structure and elicit an immune response that produces antibodies with binding specificity to the antigen.
The term “self” is used herein to describe antigens or epitopes which would not be recognized or be only poorly recognized by the B-cell receptors of a wild type member of the host species, by virtue of being included among the substances which are normally biosynthesized by the host species, or to which the host species is normally exposed. Such substances induce tolerance of the host immune system and the host is said to be “tolerized” to the substances.
The vertebrate immune system is able to discriminate between self-antigens and foreign antigens, mounting an antibody-mediated immune response to the latter and not the former. The antibody response is mediated by the B cells. Variable-region gene rearrangements occur in an ordered sequence during B-cell maturation in the bone marrow. At the end of this process, each B cell contains a single, functional variable-region DNA sequence encoding an immunoglobulin heavy chain and a single, functional variable-region DNA sequence encoding an immunoglobulin light chain. This process leads to the generation of mature, immunocompetent B cells each of which is antigenically committed to a single epitope. In a process that is not yet understood, immunologic tolerance to “self” components is accomplished by the selective ablation of B cells with variable regions that are antigenically committed to self-epitopes. This self-tolerance precludes production of antibodies specific for antigens or epitopes that are synthesized by a host vertebrate. Thus, only antigens that contain epitopes which are recognized as foreign by the host can be used to generate antibodies.
When the self and foreign epitopes are structurally similar, or “homologous”, the host immune response is weaker; thus it is virtually impossible to obtain antibodies with high affinity to such epitopes. As a result, it is extremely difficult to generate antibodies to highly conserved domains of proteins (e.g. N-CAM, cytokines, and immunoglobulins), because animals that share the conserved domains fail to recognize them as foreign. While antibodies to self-antigens are produced as a result of certain autoimmune diseases, these antibodies have binding specificities to a highly restricted set of self-antigens which cannot be manipulated artificially and generally have low binding affinities. Thus, animals with autoimmune diseases are not widely useful in the production of antibodies with binding specificity to self-antigens.
In mice, allogeneic differences between strains have allowed the production of mouse anti-mouse antibodies specific for proteins of which such allogeneic differences have been produced. Kessler et al. (1979) J. Immunol. 123:2772–2778; Reif and Allen (1964) J. Exp. Med. 120:413–433; Marshak-Rothstein (1979) J. Immunol. 122:2491–2497; and Oi and Herzenberg (1979) Molec. Immunol. 16:1005–1017. Initially, polyclonal antisera and then monoclonal antibodies specific for T cell surface proteins and mouse IgD antibodies were obtained in this manner. These antibodies, however, only recognize the gene product of particular mouse strains. These antibodies can only recognize those/epitopes/which are not structurally homologous to the self-antigens of the antibody-producing host. Additionally, the epitopes against which these antibodies can be obtained are limited to the differences between the strains and availability of allotypic strains themselves and thus have little practical utility.
Numerous methods have been formulated to analyze and sort populations of cells including, but not limited to, fluorescence activated cell sorting (FACS), magnetic separation (using magnetic bead-conjugated antibodies) and other methods reliant upon antibody affinity to particular cell surface proteins known as “markers”. Such approaches to cell analysis and separation are especially useful for the determination of cell lineages, the isolation of cells which are capable of synthesizing a particular product, and the treatment of various disease conditions with specific cell types. For example, highly purified hematopoietic stem cells are essential for hematopoietic engraftment including, but not limited to, cancer patients and transplantation of other organs in association with hematopoietic engraftment. Isolated cell populations are also important targets for gene therapy in the treatment of genetic disorders, AIDS and various forms of cancer. Thus, there have been numerous efforts made toward isolating particular varieties of cells in substantially pure or pure form. In instances such as isolation of stem cells, efficient purification of cells of such low concentration in the body requires antibodies which recognize and bind to stem cell specific markers with high specificity. Such antibodies are difficult to obtain due to the homology between human and murine stem cell markers.